【作者】 张长伟；

【导师】 何光华；

【作者基本信息】 西南大学 ， 作物遗传育种， 2011， 博士

【摘要】 水稻是我国也是世界的主要粮食作物,稻瘟病和纹枯病的严重发生是影响其产量和品质的重要因素。生产实践证明：选育和合理栽培抗病品种是防治水稻稻瘟病和纹枯病的最经济、有效措施。因此,多年来人们运用常规育种、杂交育种技术培育出一系列抗病水稻品种,在生产上推广应用发挥了巨大的社会和经济效益。但由于病原菌遗传多样性、抗病基因匮乏、育种周期长等因素,使稻瘟病和纹枯病抗病育种成效大大降低。为提高水稻抗病育种成效,人们采用传统育种技术与组织培养、分子标记辅助育种相结合选育抗病品种,证明能缩短育种周期和提高育种效率,克服了传统育种的一些主要缺点,但育种中仍存在一些难以克服的局限,如抗病基因的缺乏和数量的不足,常使其研究举步维艰,育种成效难以彰显。而转基因技术能弥补这些育种技术的不足,已成为目前丰富水稻抗病种质资源,提高水稻抗病育种成效的有效途径之一。本实验室采用根癌农杆菌介导法将苦瓜几丁质酶基因McCHIT1、苜蓿防御素基因alfAFP及其双价基因成功地导入水稻恢复系缙恢35中,通过抗病性鉴定、GUS和PCR检测,得到了一批对稻瘟病、纹枯病抗性明显增强的转alfAFP基因、McCHITl基因、McCHIT1-alfAFP双价基因植株后代。为进一步了解这些转基因水稻植株后代对稻瘟病和纹枯病抗性的遗传变异特性,挖掘其育种利用价值,本研究从T3代起逐代进行稻瘟病和纹枯病抗性鉴定和筛选。对获得的稻瘟病和纹枯病抗性优良转基因稳定株系,一方面进行稻瘟病菌抗谱测定和主要农艺性状考察,另一方面对其所配杂交水稻组合进行稻瘟病抗性鉴定和主要农艺性状考察,以期筛选出稻瘟病抗性、纹枯病抗性和其他农艺性状优良的转基因恢复系材料,为探索广谱抗病育种新途径提供参考。主要的研究结果如下：1、转McCHIT1基因、alfAFP基因及其双价基因水稻各世代的抗病性变异结合本实验室前期的研究可见,转McCHITl基因、alfAFP基因及其双价基因McCHIT1-alfAFP基因水稻各世代的抗病性变异有基本相同的趋势,即T2代以前的株系在稻瘟病和纹枯病抗性遗传上极不稳定,不同株系间以及同一抗病性优良株系的不同单株间存在较大的抗病性差异；T3代大多数株系的抗病性基本稳定,少部分株系属杂合株系,单株间抗病性仍存在较大的强弱分化；T3代以后随着世代的增加,转基因水稻的稻瘟病和纹枯病抗性遗传逐趋稳定,通过连续多代的抗病性鉴定、GUS检测和优良抗病株系的筛选,至T5代可筛选出抗病性优良的各类型转基因水稻稳定株系。2、稻瘟病抗性优良转McCHITl基因、alfAFP基因及其双价基因水稻稳定株系的筛选及其主要农艺性状的表现通过对T3、T4、T5代株系的稻瘟病抗性鉴定、筛选和T6代株系的稻瘟病菌抗谱测定,获得稻瘟病抗性较受体对照缙恢35极显著提高、抗谱明显拓宽的转McCHIT1基因水稻稳定株系有C36-2-1、C35-6-2、C10-7-1、C24-4-1、C24-4-2、C21-6-2和C21-3-1等7个株系,转alfAFP基因水稻稳定株系有A3-6-1、A3-6-2、A7-3-1、A7-6-1、A7-6-2、A7-8-1、A4-8-1、A10-4-1等8个株系,转McCHIT1-alfAFP基因水稻稳定株系有AC2-10-1、AC4-2-2、AC7-3-1、AC8-5-1等4个株系。在受体对照感苗瘟、叶瘟和穗颈瘟的发病情况下,这些株系的苗瘟抗性、叶瘟抗性、穗颈瘟抗性达到或接近中抗水平。这些株系的总群抗病频率比受体对照之值(36.50%)高出15个百分点以上,与受体对照相比,多数株系增加了对ZE群生理小种的抗性,提高了对ZA.ZB.ZG.ZF等4个种群生理小种的抗性,对稻瘟病菌表现出比较广谱的抗性。采用“逐代增加选择压,抗中选抗”的方法,可筛选出稻瘟病抗性优良、抗谱明显拓宽的转基因稳定株系。农艺性状考察表明,各稻瘟病抗性优良转基因水稻株系的主要农艺性状已趋稳定,但与受体对照相比,不同类型转基因株系的主要农艺性状均发生了不同程度的变异。转McCHIT1基因水稻株系与受体对照相比变异较大,主要趋势为,转基因株系的播始历期较短,植株较矮,单株有效穗数较多,穗长较短,每穗粒数较多,穗着粒较密,结实率较低,千粒重较小,而单株实粒重则趋势不明,较高、较低的变化均有,其中,以播始历期、株高、结实率和千粒重等4个性状的差异较为显著而普遍。转alfAFP基因水稻株系与受体对照相比,除单株有效穗数差异不显著,播始历期显著较长、显著较短均有,植株株高显著较高、显著较矮均有外,在其他性状上的主要趋势与转McCHT1基因水稻株系的一致,其中,在播始历期、结实率、株高等3个性状上存在较为广泛而显著的差异。转McCHIT1-alfAFP基因水稻株系与受体对照相比,穗粒数、穗着粒密度等2个性状差异不显著,播始历期较长、较短均有,单株有效穗数较多,单株粒重较大,在结实率和千粒重等2性状上与转McCHIT1-alfAFP基因水稻株系的一致,其中,以千粒重、播始历期、株高、穗长的差异较为显著而普遍。结合水稻恢复系选育的要求,综合比较,C36-2-1、C21-6-2、C21-3-1、A-7-3-1、A7-8-1、AC2-10-1和AC4-2-2等7个株系是综合农艺性状较好的抗稻瘟病转基因株系。3、稻瘟病抗性优良转McCHIT1基因、alfAFP基因及其双价基因水稻稳定株系所配组合的稻瘟病抗性及其主要农艺性状表现转基因株系所配组合的稻瘟病抗性鉴定和农艺性状考察结果表明,各抗病性优良转基因株系所配杂交水稻组合的稻瘟病抗性极显著优于受体对照和感病转基因株系所配组合,其优良抗病性能较好地传递给F1代,但转alfAFP基因株系的优良抗病性传递能力相对较好,所配组合的穗颈瘟抗性较强,达到或接近中抗水平。各抗病性优良转基因株系所配杂交水稻组合的主要农艺性状已趋稳定,与受体对照所配组合相比,除个别株系所配组合在个别或少数考察性状上与受体对照所配组合存在显著或极显著差异外,多数抗病性优良转基因株系所配组合在主要农艺性状上与受体对照所配组合相当。这与抗病性优良转基因株系的主要农艺性状与受体对照有较大差异不一致。综合比较,C21-6-2、C21-3-1、C36-2-1、A7-3-1、A7-8-1、AC2-10-1、AC4-2-2和AC7-3-1等8个抗病性优良转基因株系与水稻不育系II-32A所配组合是丰抗结合较优的转基因杂交水稻组合。4、纹枯病优良抗性转McCHITl基因、alfAFP基因及其双价基因水稻稳定株系的筛选通过对T3、T4、T5代株系的纹枯病抗性鉴定、筛选,获得了C36-2-1、C35-6-2、C10-7-1、C24-4-1、C10-2-1、C24-2-1、C24-2-2、C21-13-2等8个纹枯病抗性优良(病情指数值低于45%以下)的转McCHIT1基因株系,其中,C36-2-1、C10-7-1、C21-6-2、C21-3-1等4个株系属纹枯病和稻瘟病抗性均优良的转McCHIT1基因株系；获得了A3-6-1、A3-6-2、A7-6-1、A4-8-1、A10-4-1、A3-2-2、A2-9-3等7个纹枯病抗性优良(病情指数值低于45%以下)的转alfAFP基因株系,其中,A3-6-1、A3-6-2、A7-6-1、A4-8-1、A10-4-1等5个株系属纹枯病和稻瘟病抗性均优良的转alfAFP基因株系；获得了AC2-10-1、AC4-2-2、AC7-3-1、AC3-2-1、AC6-2-2等5个纹枯病抗性优良(病情指数值低于45%以下)的转McCHIT1-alfAFP基因株系,其中,AC2-10-1、AC4-2-2、AC7-3-1等3个株系属纹枯病和稻瘟病抗性均优良的转McCHIT1-alfAFP基因株系。5不同转McCHIT1基因、alfAFP基因及其双价基因水稻稳定株系的抗病性强弱分化的原因分析稻瘟病菌抗谱分析表明,稻瘟病抗性优良的7个转McCHIT1基因株系、8个转alfAFP基因株系和4个转McCHIT1-alfAFP基因株系的稻瘟病抗性显著或极显著优于受体对照和其他转基因株系的主要原因是,其对稻瘟病菌总群的抗谱明显较宽,特别是对优势种群B群和重要种群A群的生理小种具有较高的抗病频率。几丁质酶活性分析表明,抗病性优良转McCHIT1基因株系在接种纹枯病菌前后的总几丁质酶活性显著或极显著高于受体对照和感病转基因株系的总几丁质酶活性,其接种前后1d的总几丁质酶活性是受体对照的3.4-4.2倍,是感病转基因株系2.1-2.7倍,接种2d后随着受体对照的总几丁质酶活性的快速提高,抗病性优良转基因株系的总几丁质酶活性较受体对照的倍率有所下降,为1.8-2.9倍,但抗病性优良转基因株系的总几丁质酶活性较接种2d前有所提高,但不显著,说明抗病性优良转基因株系的McCHIT1基因得到了较强程度的组成型表达,同时内源几丁质酶基因也能得到一定程度的诱导性表达。因此,感病转基因株系的McCHIT1基因未能高水平的组成型表达,是其抗病性显著弱于抗病性优良转基因水稻株系的原因,反之,抗病性优良转基因水稻株系的抗病性显著或极显著高于受体对照和其他转基因株系的生理原因是其McCHIT1基因得到了高水平的组成型表达。更多还原

【Abstract】 Rice is an important food crop all over the world. Rice blast and sheath blight are important diseases influencing rice yield and quality. It has been approved that breeding and planting resistant cultivars is the most economical and effective way to control rice blast and sheath blight.To date, a series of rice varieties with resistance to disease were bred by various conventional methods, which exit enormous social and economic function in agricultural production. However, as the result of genetic diversity of nosophyte, absence of resistance genes and long breeding circle in traditional methods, the breeding effect for disease resistance was lower to a great degree. To improve breeding effect in rice, conventional breeding methods connected with tissue culture and molecular marker assisted selection technique were applied to breeding for disease resistance. Though overcoming large amount of defects in traditional breeding, some issues were still existed, i.e. insufficient genes for disease resistance. Just the transgenic technique can remedy the deficiency, and has become an effective approach in breeding for disease resistance.In our laboratory, disease resistance genes McCHIT from balsam pear (Momordica charantia L.), alfAFP from clover (Medicago sativa L.) and their bivalent gene (McCHIT1-alfAFP) were transferred to rice restorer line Jinhui35 by Agrobacterium-mediated transformation method. By detection of disease resistance and GUS as well as PCR, we obtained a set of transgenic rice plants harboring McCHITl, alfAFP and McCHIT1-alfAFP gene whose descendants obviously strong resistance to blast and sheath blight. For further understanding the inherited character of the genes in transgenic plant descendant and screening stable plant descendant with blast and sheath blight resistance, estimating and screening resistance to blast and sheath blight were done from T3 in the study. Once the stable and outstanding transgenic plants with blast and sheath blight resistance were obtained, on the one hand, their resistance spectrum and main agronomic traits were valued, on the other hand, hybrid rice F1 crossed by sterile lines with them were appraised by the same targets involved above. We expect screening excellent transgenic rice restorer and exploring a novel route for broad-spectrum disease resistance. The main results were as follows:1. Genetic variation of disease resistance for rice transgenic plants harboring McCHITl,alfAFP and McCHIT1-alfAFP in various generationsTogether with the prophase study results, there were same trends basically in disease resistance of different generations for transgenic plants harboring McCHITl, alfAFP and McCHIT1-alfAFP gene. At the low generation, especially before T2 generation, resistance inheritance for blast and sheath blight were unstable, exhibiting larger difference among various lines even plants within the same line. At T3 generation, disease resistance was stable basically for most lines, only unstable for a few heterozygosis plants in which resistance still exist polarization among plants. With the increasing of generation after T3, transgenic lines to resistance were gradually stable. In T5 generation, different types of stable transgenic plants with excellent disease resistance were screened out by successive detection of disease resistance and GUS test.2. Stable and noticeable M. grisea-resistance transgenic rice lines harboring McCHITl, alfAFP and McCHITl-alfAFP gene and its major agronomic characteristicsBased on the analysis of resistance to rice blast in T3, T4 and T5 generations and on identification of resistance spectrum incubated by M. grisea in T6 generation. transgenic plant lines increasing resistance and extended resistance-spectrum to rice blast were screened as follows:seven McCHITl-transgenic rice lines i.e. C36-2-1, C35-6-2, C10-7-1, C24-4-1, C24-4-2, C21-6-2 and C21-3-1, eight alfAFP-transforming rice lines i.e. A3-6-1, A3-6-2, A7-3-1, A7-6-1, A7-6-2, A7-8-1, A4-8-1 and A10-4-1, and four McCHIT1-alfAFP-transforming rice lines i.e. AC2-10-1, AC4-2-2, AC7-3-1 and AC8-5-1. These lines demonstrated the mid-level resistance to leaf and neck blast as well as resistance in seedling stage to leaf blast or at least closely. In detail, when the wild-type control was highly sensitive to M. grisea was of a disease, transgenic plants were slightly destroyed no matter leaf and neck blast or leaf blast at seedling stage. The value of disease-resisted frequency increased more than 15% in transgenic plant lines compared with the control of wild type 36.50%, and the majority of transgenic rice acquired the resistance to ZE groups and enhanced the resisted-level to ZA, ZB, ZG and ZF groups by identification of broad-spectrum resistance. Consistently, excellent transgenic rice lines could be found based on the method of selection pressure increased from generation to generation and mid-resistance lines kept.After self-pollination of generations, the key agronomic characteristics have inherited inherited stably in despite of the distinct differences between transgenic lines. In the case of McCHITl gene over-expression, compared with the wild type, the transgenic lines mainly showed the shorten life cycle, dwarfism, decreased effective panicle number per plant, reduced panicle length, increased grain number and seed density per panicle, poorly seed setting, lower weight of 1000-seeds and filled seeds per plant, interestingly, four of which demonstrated universal and significant difference with these of the wild type, that is life cycle span, plant height, seed setting and 1000-seeds weight. As for transgenic plant alfAFP gene over-expression, effective panicle number per plant showed no significant difference between wild type and transgenic lines, life cycle showed significant longer or shorter and plant height increased or decreased significantly in the transgenic rice compared with those of wild type control, and other agronomic traits changed consistently with the plants of McCHIT1 gene over-expression, of which, life cycle, seed setting ratio and plant height were severely reconstructed. When McCHITl-alfAFP genes were transformed to rice, the progeny of transgenic lines displayed no significantly difference in grain number and seed density per panicle, other agronomic characteristics such as life cycle, effective panicle number per plant and seed weight per plant were significantly negatively or dominantly affected in some off-springs of transgenic plant, and the changing of seed setting ratio and 1000-seeds weight was consistent with rice plant harboring McCHITl and alfAFP respectively. For all agronomic traits,1000-seeds weight, life cycle, plant height, panicle length and seed setting ratio were wildly and severely influenced at the McCHIT1-alfAFP-transforming rice. Given the breeding of rice restorer lines, seven lines C36-2-1, C21-6-2, C21-3-1, A-7-3-1, A7-8-1, AC2-10-1 and AC4-2-2 were excellent transgenic plant lines against to rice blast.3. M. grisea resistance and agronomic traits analysis of hybrid rice varieties crossed with screened transgenic lines as male, harboring McCHITl, alfAFP and McCHITl-alfAFP gene respectively.Hybrid combinations were acquired by crossing predominant transgenic lines as males, rice blast resistances were performed and agronomic characteristics were identified subsequently. The results showed as follows:M. grisea resistances were remarkably increased in hybrid combinations with the male of M. grisea-resisted transgenic lines than in those with wild type control or M. grisea-unresisted transgenic lines as male, particularly for alfAFP over-expression lines, the combinations were nearly coincided with transgenic line in neck-blast resistance, revealed mid-resistance level at least closely, which suggested the resistance could be inherited to F1 generations dominantly in the transgenic rice. In essence, the main agronomic traits were inherited stably and equivalent to the wild-type control in F1 generations, significant discrepancies were only detected in single or several traits of the combinations crossed with exceptive transgenic lines by comparison with these of wild type, this result disaccord with the multiple mutations of major agronomic characteristics in transgenic lines. Taken together, the hybrid combinations with excellent productive traits and higher resistance to M. grisea were bred by crossing CMS line II-32A with transgenic lines of C21-6-2, C21-3-1, C36-2-1, A7-3-1, A7-8-1, AC2-10-1, AC4-2-2 and AC7-3-1 respectively.4. Identification of rice sheath blight resistance transgenic lines harboring McCHIT1, alfAFP and McCHIT1-alfAFP gene respectivelyEight plant lines were screened against to rice sheath blight (the value of disease index less than 45%) by T3, T4 and T5 generation identification of McCHITl-transforming rice, which were C36-2-1, C35-6-2, C10-7-1, C24-4-1, C10-2-1, C24-2-1, C24-2-2 and C21-13-2, of which, four lines i.e. C36-2-1, C10-7-1, C21-6-2, C21-3-1 showed predominant resistance to both rice blast and rice sheath blight. By over-expression of alfAFP gene, seven transgenic lines acquired rice sheath blight resistance (the value of disease index less than 45%), they were A3-6-1, A3-6-2, A7-6-1, A4-8-1, A10-4-1, A3-2-2 and A2-9-3, comparably, five of which showed double resistance including AC2-10-1, AC4-2-2, AC7-3-1, AC3-2-1 and AC6-2-2. As for McCHIT1-alfAFP genes, only five transgenic lines i.e. AC2-10-1, AC4-2-2, AC7-3-1, AC3-2-1 and AC6-2-2 exhibited the value of disease index less than 45% by identification of rice sheath blight resistance, of which, AC2-10-1, AC4-2-2 and AC7-3-1 revealed double resistance to both rice blast and rice sheath blight.5. Analysis of the differentiation of resistance strength to M. grisea and R.solani. among different stable transgenic lines harboring McCHITl,alfAFP and McCHIT1-alfAFP gene respectively.The analysis of grisea resistance spectrum showed that 7 McCHITl-transforming rice lines have excellent blast resistance, the blast resistance of 8 alfAFP-transforming lines and 4 McCHIT1-alfAFP-transforming lines represent a highly significant compared with control and other transgenic lines. The mainly reason is that the anti-spectrum of the total population of magnaporthe grisea are significantly wider, especially the physiological race of dominant group B and important group A have higher frequency of resistance. The analysis of chitinase activity showed that the chitinase activity of good disease resistance in McCHITl-transforming lines before and after inoculation Rhizoctonia solani were more significant than those of control control and other transgenic lines. The total chitinase activity were 3.4 to 4.2 times as much as the receptor at 1 d after inoculation and 2.1 to 2.7 times as much as susceptible transgenic plants. With the rapidly increase of the total chitinase activity of receptor at 2d after inoculation. The total chitinase activity of good disease resistance of transgenic decreased compared with the times of control were 1.8 to 2.9. But the total chitinase activity increased more than 2 d before, which showed that the McCHIT-transforming plants with excellent disease resistance have strong constitutive expression. Meanwhile, endogenous chitinase gene can be induced to express. Susceptible McCHIT1-transforming lines have not been a high level of constitutive expression, which is the reason why the disease resistance of susceptible transgenic plants was significantly weaker than excellent disease resistance of transgenic rice lines. Otherwise, the disease resistance of excellent disease resistance of transgenic rice was significantly higher than those of control or the other transgenic lines. The reason of physiological reasons is that McCHITl has a higher constitutive expression in McCHIT-transforming rice with excellent disease resistance than in the susceptible transgenic lines.更多还原